Molecular Imaging and Biology. 2013 Oct 1; 15(5):625-32

Assessment of the novel estrogen receptor PET tracer 4-fluoro-11β-methoxy-16α-[18F]fluoroestradiol (4FMFES) by PET imaging in a breast cancer murine model.

Paquette M, Phoenix S, Ouellet R, Langlois R, van Lier JE, Turcotte ÉE, Bénard F*, Lecomte R

Sherbrooke Molecular Imaging Center of CRCHUS, Department of Nuclear Medicine and Radiobiology, Université de Sherbrooke, Sherbrooke, QC, Canada

*BC Cancer Agency Research Center, Department of Radiology, University of British Columbia, Vancouver, BC, Canada.



Purpose: The aim of this study was to compare the in vivo stability, uptake, and positron emission tomography (PET) imaging performance of a novel estrogen receptor PET tracer, 4-fluoro-11β-methoxy-16α-[18F]fluoroestradiol (4FMFES), with 16α-[18F]-fluoroestradiol (FES).

Procedures: MC7-L1 and MC4-L2 (ER+) cell lines and their ERα-knockdown variants (ERαKD) were implanted subcutaneously in Balb/c mice. After 21 days, mice were imaged using either FES or 4FMFES. One hour post-injection, static images were acquired for 30 min and the tumor %ID/g uptake values were derived. Biodistribution data were also obtained 1 h following the injection of either FES or 4FMFES. Blood samples were taken at different times and analyzed on thin-layer chromatography to quantify the presence of radiometabolites for each radiotracer. To assess specific targeting to the estrogen receptors, mice bearing only ER+ tumors were treated with the competitive ER inhibitor fulvestrant 48 h prior to imaging with 4FMFES.

Results: Metabolic stability was found to be similar for both tracers in mice. Both FES and 4FMFES differentiated ER+ tumors from ERαKD tumors in biodistribution and PET imaging studies. 4FMFES achieved a significantly higher %ID/g uptake in ER+ tumors and MC4-L2 ERαKD tumors than FES in the PET imaging studies. Also, tumor-to-background ratio was higher in ER+ tumors using 4FMFES compared to FES. Dissection data showed a significantly higher %ID/g in all tested cell lines and ER-rich tissues using 4FMFES versus FES. Fulvestrant-treated mice had either low or undetectable tumor uptake.

Conclusion: In a tumor-bearing mouse model, 4FMFES achieves better specific tumor uptake and better contrast than FES, making it a promising candidate for ER imaging.

Keywords: Receptor imaging, Estrogen receptor α, Breast cancer, Knockdown tumor model, Small animal PET

PMID: 23619898



Knowledge of the Estrogen Receptor (ER) status strongly influences prognosis and course of treatment in breast cancer management. ER level of biopsied tissue is routinely being assessed by immunohistochemistry (IHC) in clinics. However, biopsy samples can be hard to obtain for small metastatic foci, and it is known that ER status in metastatic disease may be different from the primary lesion. Moreover, 20% to 40% of patients with an ER-positive (ER+) tumor initially become ER-negative (ER-) in case of recurrence.

Efforts have been made to assess ER status non-invasively, mainly using [18F]-16α-fluoroestradiol (FES) PET imaging. While FES uptake correlated well with ER status and ER IHC index, its rapid metabolic processing and elimination by the liver lead to increased unbound low-affinity metabolites in the blood, resulting in poor contrast and low overall image quality.

In order to improve ER PET imaging, many new estradiol-derived compounds were designed and evaluated in the last few decades, with little or no success. Attempts in our center to improve the properties of 11β-methoxy-FES derivatives lead, amongst other compounds, to the synthesis and characterization of 4-fluoro-11β-methoxy-16α-[18F]fluoroestradiol (4FMFES). While 4FMFES exhibited a 4-fold decrease in in vitro ER binding, it achieved the best in vivo specificity, along with a better tumor-to-nontarget ratio in dissected ER+ mammary tumor-bearing female mice.

To further evaluate the PET imaging potential of 4FMFES and to compare its performance with the well-known FES, a comparative tumor-bearing murine model was used. Each mouse was implanted with 2 different murine ER+ mammary carcinomas (MC7-L1 and MC4-L2 cell lines), along with their ERα-knockdown (ERαKD) version having a reduced ERα expression. Tumors were grown up to 4 mm in diameter prior to PET imaging and dissection experiments.

The tumor-bearing mice were imaged using a high-resolution small animal PET scanner to obtain 30-minute static images at 1 hour after injection of either 4.5 MBq FES or 10.5 MBq 4FMFES. The tumor uptake was estimated from the PET images in units of cps/µl and converted to Bq/µl. In parallel, another group of mice were dissected 1 hour post-injection of either tracers in order to correlate with PET imaging data.

Results showed a slight but significant increase in uptake and contrast using 4FMFES PET as compared to FES PET for both ER+ tumor types. As a consequence, overall image quality was improved using 4FMFES PET (Fig. 1). While the ERαKD tumor uptake assessed by biodistribution was found to be slightly higher using 4FMFES (Fig. 2a), no significant differences in uptake and contrast were observed by PET imaging between FES and 4FMFES (Figs. 2b and 2c). Both tracers allowed ER+ tumors to be easily discriminated from ERαKD tumors by uptake and contrast analysis. Unexpectedly, time-dependent blood metabolites analysis by thin-layer chromatography showed that both tracers were equally metabolized in mice (Fig. 3), in contrast with preliminary clinical data in humans, which showed a 2.5-fold increase of intact 4FMFES in blood as compared to FES, suggesting a higher resistance of 4FMFES to human metabolism. We therefore conclude that the better imaging performance of 4FMFES in mice most likely results from its intrinsic properties, while the improved metabolic profile suggests that even better results may be obtained in humans.

In summary, according to this preclinical study and previous work, 4FMFES shows great promises as an alternative to FES for optimal ER PET imaging. A phase II clinical trial comparing 4FMFES to FES PET in breast cancer patients is in progress to confirm these findings.



This project was supported by the Canadian Institutes of Health Research grant #MOP-86717 and the Canadian Breast Cancer Research Alliance grant #015388.

The Research Center of CHUS) CRCHUS is funded by Le Fonds de recherche du Québec – Santé.


Figure-1-Paquette-WBFFigure 1 : Representative coronal (left) and transaxial (right) slices centered on the tumors of either FES or 4FMFES PET images.

Created with The GIMPFigure 2 : Tumor uptake (expressed in %ID/g) as determined by biodistribution (a) and PET imaging (b), and tumor-to-background (T/B) ratio (c) measured from PET images. *p<0.05

Figure-3-Paquette-WBFFigure 3 : Average % of intact tracer remaining in the blood stream as assessed by reverse-phase thin-layer chromatography (TLC) analysis at each time point.

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